Serum amyloid A contributes to radiation-induced lung injury by activating macrophages through FPR2/Rac1/NF-κB pathway

Abstract

Patients who receive thoracic radiotherapy may suffer from radiation-induced lung injury, but the treatment options are limited as the underlying mechanisms are unclear. Using a mouse model of right thorax irradiation with fractionated doses of X-rays for three consecutive days (8 Gy/per day), this study found that the thoracic irradiation (Th-IR) induced tissue injury with aberrant infiltration of macrophages, and it significantly increased the secretion of TNF-α, IL-1β, IL-6, TGF-β1 and serum amyloid A (SAA) in mice. Interestingly, SAA could activate macrophages and then induce epithelial-mesenchymal transition (EMT) of lung epithelial cells and fibrosis progression in lung tissue. Mechanistically, SAA enhanced the transient binding of FPR2 to Rac1 protein and further activated NF-κB signaling pathway in macrophages. Inhibition of FPR2 significantly reduced pulmonary fibrosis induced by SAA administration in mice. In addition, cimetidine could reduce the level of SAA release after irradiation and attenuate the lung injury induced by SAA or Th-IR. In conclusion, our results demonstrated that SAA activated macrophages via FPR2/Rac1/NF-κB pathway and might contribute to the Th-IR induced lung injury, which may provide a new strategy to attenuate radiation-induced adverse effects during radiotherapy.

Keywords: FPR2 and NF-κB; Lung injury; Macrophages; SAA; Thoracic irradiation.

Figure 1

Th-IR induced tissue damage in the irradiated lung tissues of mice. (A) IHC staining and western blot assay for γ-H2AX proteins in lung. (B) H&E staining images and quantitative analysis of pneumonia and fibrosis of lung. Blue arrows indicate the thickened alveolar wall and green arrows indicate hemorrhage exudate. (C) IHC staining for α-SMA and Masson's trichrome staining of lung at 60 and 180 days after Th-IR. Scale bar=150 μm, magnifying scale bar=50 μm. n=6-8 for each group, ** p < 0.01, *** p < 0.001, **** p < 0.0001 compared with Sham-IR group. NS, no significant difference.

Figure 2

Th-IR affected the macrophage distribution and phenotypes in lung and cytokine expression. (A) IF staining images of macrophages in lung. (ⅰ, ⅲ) Proportion of M1/M2 macrophages over the lung tissues; (ⅱ, ⅳ) Proportion of M1/M2 macrophages over the total number of macrophages in lung. Scale bar=100 μm. (B-C) IHC staining (B) and positive scores for TGF-β1 expression (C) in lung. Scale bar=150 μm, magnifying scale bar=50 μm. (D-F) The concentrations of TGF-β1 (D) SAA (E), TNF-α, IL-1β, and IL-6 (F) in mouse serum after Th-IR. n=6-8 for each group, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 compared with Sham-IR group.

Figure 3

SAA promoted EMT progression in lung epithelial cells by activating macrophages. (A) H&E staining of lung tissues at 10 weeks after the first SAA injection. Scale bar=150 μm, magnifying scale bar=50 μm. (B-D) Expression of indicated proteins in MLE-12 cells that were treated with 5 μg/mL SAA for 0, 12, 24, and 48 h (B) or incubated with the conditioned medium of macrophages for 24 h, where the macrophages were pretreated by 0 μg/mL (C) or 5 μg/mL SAA for 0, 2, 6, and 12 h (D). (E-F) Relative proliferation level (E) and ROS level (F) of RAW264.7 macrophages treated with SAA. (G-H) Representative images (G) and cell cycle distribution (H) of RAW264.7 macrophages treated with or without SAA. (I) IF staining for iNOS (red), Arg-1 (green), and DAPI (blue) in RAW264.7 cells treated with/without SAA for 6 h. Scale bar=100 μm. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 between indicated groups.

Figure 4

SAA activated FPR2/Rac1/NF-κB pathway in RAW264.7 macrophages. (A) Expression of NF-κB/p-NF-κB protein in macrophages measured by western blot assay. (B-C) Expression of indicated proteins (B) and IF staining of F-actin (green) and Vimentin (red) (C) in MLE-12 cells incubated for 24 h with the conditioned medium of macrophages pretreated with or without NF-κB inhibitor of BAY 11-7082 (2 μM) for 12 h. Bar=50 μm. (D-E) Expressions of indicated proteins in macrophages pretreated with NSC23766 (Rac1 inhibitor) (D) and WRW4 (FPR2 inhibitor) (E) for 24 h and 2 h, respectively, before SAA treatment. (F) IF staining for F4/80 (purple), Rac1-GTP (green), and p-NF-κB p65 (red) in mouse lungs on day 1 after Th-IR and WRW4 treatment. The mice were injected intraperitoneally with FPR2 inhibitor WRW4 for 2 h before irradiation, and lung tissues were collected at 1 day after Th-IR (n=6-8 for each group). Scale bar=30 μm, magnifying scale bar=10 μm. * p < 0.05, ** p < 0.01, *** p < 0.001 between indicated groups.

Figure 5

SAA promoted protein binding of FRP2 and Rac1 in RAW264.7 macrophages. (A) Protein expression of FRP2 and Rac1-GTP in macrophages after the cells treated by SAA at different time points. (B) IF staining for FRP2 and Rac1-GTP protein in RAW264.7 macrophages. Scale bar=50 μm. (C) Online STRING analysis of protein-protein interaction among FPR2, Rac1, and Akt. (D) Co-IP assay of FRP2 and Rac1 protein in macrophages treated by SAA. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 between indicated groups.

Figure 6

CMTD alleviated lung injury induced by irradiation. The mice were given CMTD (100 mg/ kg/day) by oral gavage from the first day of Th-IR and continued to 7 days after Th-IR. (A) H&E staining of lung and quantitative analysis of injury of lung at day 3 and day 60. (B) Masson trichrome staining and IHC staining for α-SMA of lung at day 60 after Th-IR. (C) IHC staining for TGF-β1 of lung at day 60 after Th-IR. Scale bar=150 μm, magnifying scale bar=50 μm. (D) IF staining for F4/80 (purple), iNOS (green), CD206 (red), and DAPI (blue) in mouse lung at day 60 after Th-IR. Scale bar=100 μm. n=6-8 for each group, ** p < 0.01, *** p < 0.001, **** p < 0.0001 between indicated groups. NS, no significant difference.

Figure 7

Inhibitor of FPR2 or CMTD alleviated lung injury induced by SAA. (A) Schematic flow of WRW4 or CMTD treatment in SAA-indued lung injury mouse model. The mice were given an FPR2 inhibitor (WRW4, 1.8 mg/kg) via intraperitoneal injection or CMTD (100 mg/kg/day) by gavage at 2 h before SAA treatment. (B-D) H&E staining (B) and IHC staining for TGF-β1(C) and α-SMA (D) of lung tissue at 10 weeks after the first SAA injection. Scale bar=150 μm, magnifying scale bar=50 μm. (E) IF staining for F4/80 (purple), iNOS (green), CD206 (red), and DAPI (blue) in mouse lungs treated with/without SAA at 10 weeks after the first SAA injection. Scale bar=100 μm. n=6-8 for each group, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 between indicated groups.

Figure 8

Schematic mechanism of thoracic irradiation inducing lung injury via the SAA/FRP2/Rac1/NF-κB pathway. Fractionated irradiation of the right thorax triggers the cytokine production and up-regulates serum SAA level, which induces EMT and fibrosis process of lung epithelial cells via activation of the FPR2/Rac1/NF-κB pathway in macrophages.